Abstract

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

Abstract

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages.

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